Avian Coronavirus Infectious Bronchitis Virus. Dr. Mark W. Jackwood University of Georgia Athens, GA USA

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1 Avian Coronavirus Infectious Bronchitis Virus Dr. Mark W. Jackwood University of Georgia Athens, GA USA

2 Infectious bronchitis virus is a highly contagious avian coronavirus Multiple serotypes and variants of the virus exist

3 Infectious bronchitis virus is found worldwide Control is primarily through the use of modified live vaccines but The disease is extremely difficult to control because different serotypes and variants do not cross protect

The Disease Mild upper-respiratory tract disease

eyes, nasal mucus, and swollen sinuses Decreased egg

can cause lesions in the kidneys (Nephritis).

4 The Disease Mild upper-respiratory tract disease Sneezing, tracheal rales, gasping, coughing, watery eyes, nasal mucus, and swollen sinuses Decreased egg quality and production in layer birds Some strains can cause lesions in the kidneys (Nephritis). Morbidity ~100% Mortality varies with complicating infections (E. coli)

5 IBV History of Serotypes IBV was first recognized circa 1930, and for about 15 years Mass was the only known serotype. In 1956 Jungherr et al. described a new serotype of IBV from Connecticut and designated it Conn. Now there are many recognized serotypes and countless variant viruses

6 The first IBV vaccines Drs. Beaudette and Hudson in 1937 discovered that IBV could be grown in embryonated eggs Dr. van Roeckel reported on a planned exposure to control IBV in 1942 The first USA vaccine was developed in the 1950s using the van Roeckel M-41 strain which is a Mass type virus isolated in Massachusetts in 1941 The M-41 strain is the parent strain for most of the Mass type vaccines used in the USA

7 IBV is a coronavirus Single stranded RNA genome 28+ kb (largest known) 4 structural proteins (Spike, Envelope, Membrane, Nucleocapsid) Spike glycoprotein on the surface of the virus structurally it has two subunits S1 & S2 Antigenic diversity is due to variability in spike

8 IBV- Serotypes Spike mediates virus-cell attachment and entry into the cell Spikes have several regions on their surface (epitopes) that induce neutralizing antibodies. Epitopes on spike are unique for each IBV type Neutralizing antibodies bind to spike and prevent infection

9 IBV Diagnosis Virus isolation and rising antibody titers are the traditional methods for making a definitive diagnosis Mabs (Mass, Conn, Ark) Genotyping Collection of samples (timing is important) Tracheal swabs (choanal cleft) and/or cecal tonsils Placement of sentinel chickens

10 Sample Submission Live birds Whole tracheas Tracheal or choanal swabs in PBS or viral transport media (25 samples/flock, pool 5 swabs/tube) International Swabs in a 1:1 solution of PBS and molecular biology grade buffered phenol (ph 4.5) Allantoic fluid in a 1:1 solution of fluid to phenol Swabs or tissue spotted onto FTA cards

11 Finders Technology Associates (FTA) cards Filter paper for inactivation of infectious agents Cotton-based cellulose membrane Contains lyophilized chemicals that inactivate most bacteria and viruses. Immobilizes and stabilizes nucleic acid (RNA and DNA) for subsequent PCR testing Samples can be safely transported via regular Postal Service. -With the proper permit samples can be submitted from outside the USA

Diagnosis- Serology ELISA Not type specific Detects IgG antibodies against the virus Can be positive by 2 weeks

(HI) Type specific almost IBV treated with neuraminidase will agglutinate RBCs HA antigen for Ark, Mass, Conn,

Cross reactivity prevents definitive type specific results Virus neutralization (embryos, cell culture or organ

12 Diagnosis- Serology ELISA Not type specific Detects IgG antibodies against the virus Can be positive by 2 weeks PI Agar gel precipitin (AGP) test Detects IgM Can be positive by 7 or 8 days PI Hemagglutination inhibition (HI) Type specific almost IBV treated with neuraminidase will agglutinate RBCs HA antigen for Ark, Mass, Conn, JMK, and DE072 Serum reacted with each antigen and an inhibition titer is determined. Cross reactivity prevents definitive type specific results Virus neutralization (embryos, cell culture or organ culture) Type specific Detects IgG approximately 2 weeks PI Two methods Alpha- constant serum diluted virus Beta- diluted serum constant virus

13 Diagnosis- Virus Isolation 9-11 day old embryonating eggs Inoculate CAS Typical lesions in embryos are: stunting, hemorrhagic, curled embryos, with ureate deposits in the kidneys. Chicken embryo tracheal organ culture Primary chicken kidney cells Susceptible chicks

14 Molecular Diagnosis Molecular detection- real time RT-PCR Molecular identification- Genetic typing

15 Real Time RT-PCR detection of IBV Used to rapidly screen field samples for IBV Extremely rapid (20 minute test) Does not identify IBV type Determines the amount of virus in the sample Real time RT-PCR is a standard RT-PCR reaction that includes a fluorescent dye or labeled probe used to detect amplified DNA as it is being made

16 Real Time RT-PCR +/- Screening tool L 1a 1ab S1 S2 E M N AAAAA 29 CT 25 Cycle threshold (CT) value is the point where fluorescence in the reaction crosses a minimum value considered to be positive

17 Genetic basis for molecular typing of IBV L 1a 1ab S1 S2 E M N AAAAA Mass Ark Viral RNA mrna Protein Viral RNA mrna Protein A change in the spike gene can translate into a change in the amino acid sequence of spike G C C G

18 Sequencing to identify IBV type L 1a 1b S1 S2 E M N AAAAA RT-PCR Amplify Spike Spike 1720 bp HVR 450 bp Run Sequencing reaction BLAST analysis Basic Local Alignment Search Tool

Sequencing Analysis to Identify IBV type Basic Local Alignment Search Tool (BLAST) Phylogenetic tree Score E Sequences producing significant alignments: (Bits) Value gi 50082761 gb AY641576.

19 Sequencing Analysis to Identify IBV type Basic Local Alignment Search Tool (BLAST) Phylogenetic tree Score E Sequences producing significant alignments: (Bits) Value gi gb AY Avian infectious bronchitis 196 8e-48 gi gb AF AF Avian infectious 196 8e-48 gi gb AY Avian infectious bronchitis 196 8e-48 gi emb X COIBVSP1 Infectious bronchitis 196 8e-48 gi gb L IBBSPIKE Infectious bronchitis 196 8e-48 gi gb M IBAPEPE Avian infectious bronchiti 196 8e-48

20 General IBV Epidemiology The virus is evolving- New serotypes (variants) continue to arise New IBV like viruses emerge in other species (turkeys, pheasants, pea fowl). Some IBV types remain geographically restricted whereas others do not.

21 Phylogenetic analysis of IBV around the world Phylogenetic analysis provides a measure of relatedness between isolates. The closer the relatedness the more likely they will cross-protect

22 IBV types- North America GA/124/11 (similar to CA/1735/04) most recent variant in the SE USA Georgia 07 & Georgia 08 -variants in the SE USA related to CAV viruses Georgia 98 (GA98)- similar to DE072 (Vaccine available, Lee et al. Avian Dis 2001) Arkansas- the most frequently isolated virus Genetic drift is occurring in the Ark viruses (Jackwood et al. Avian Dis 2005) California variant viruses- 3 groups recognized CAV/CV56b/91 (Schikora et al. Arch. Virol. 2003) CAL/CAL99/99 (Mondal et al. Virology 2004 & Alvarado, et al. Avian Dis. 2003) CAV/1737/04 (Jackwood, et al. Avian Dis. 2007) Recent variants identified in 2004/2005

23 Amino Acid Substitutions (x100) Egypt/D274/D/89 UK/B/D274/84 Netherlands/B/D207/84 Russia/D274/15/09 UK/E/D3896/84 Brazil/USP-01/05 Slovenia/Italy02/257/09 Ukraine/Italy02/01/07 Italy/Italy-02/497/02 Japan/Gray/ON/74 USA/Gray/Gray/60 Mexico/BL56/19/UNAM/08 Mexico/47/UNAM/01 USA/PA/171/99 USA/PA/Wolgemuth/98 USA/CAV/CAV56b/91 Korea/KI/K620/02 Kazakhstan/Ark/KZ14/07 USA/Ark/ArkDPI/81 USA/CAL99/CAL99/99 USA/CA/557/03 Australia/Subgroup1/N1/62 China/N1-62/JAAS/04 USA/GA08/GA08/08 Belgian/B1648/96 Japan/JP-I/C78/99 Egypt/IS720/Beni-Seuf/01 Iraq/IS720/Sul/01/09 Israel/IS720/720/99 Israel/IS720/885/00 Israel/Variant2/98 China/LDL/Q1/98 Italy/Q1/11VIR3141-7/11 Taiwan/Q1/3374/05 India/793B/NMK/72/IVRI/10 UK/793B/4-91/91 China/793B/Sichuan/06 France/CR88121/88 Israel/793B/Variant 1/96 Iran/793B/19/08 India/744-AD/04 Russia/Mass/13/09 Japan/Mass/Nerima/00 Mexico/Mass/4/UNAM/08 Iran/Mass/17/00 Mass/H120/55 Thailand/Mass/THA320352/09 Egypt/Mass/F/03 India/Mass/16-V-AD/07 Korea/Mass/K446/01 Mass/Mass41/41 Mexico/Conn/43/UNAM/08 USA/Conn/Conn46/51 Malaysia/V9/04 Malaysia/MH5365/95 Japan/JP-II/TM86/02 USA/CA/1737/04 USA/GA07/GA07/07 India/PDRC/Pune/9/99 Korea/LX4/KII/K154/05 Spain/QX-like/La/116/09 China/LX4/QX/99 Thailand/Group2/QX-l#76FC24 Korea/KM91/91 China/LDT3/03 Korea/LDL/KIII/K147/10 Thailand/Group1/THA90151/08 China/BJ/97 China/LHLJ/95I China/LSC/99I Australia/Subgroup3/N1/03 USA/DE/DE072/92 USA/GA98/0470/98 Mexico/07484/98 Netherlands/D1466/81 Australia/Subgroup2/N1/88 Mass Conn Ark DE GA08 CAV/Cal99 GA07 PA-Nephro.

24 IBV Types- South America Brazil- BR1, BR2, BR3 genetic types % of isolates are variants and 98% of those are related to 4/91 Venezuela- one variant virus identified by RFLP, others probably exist Chile- Q1 IBV Colombia- Q1 IBV- Collaboration: Instituto Colombiano Agropecuario (ICA), Federacion Nacional de Avicultores de Colombia (FENAVI) and the University of Georgia, PDRC 4 genetic groups not related to Mass, Conn or Ark vaccines. Mass and Conn molecular types also identified. Peru- several variant viruses identified not related to the Massachusetts serotype.

25 Q1 and QX Phylogenetic Tree Ark Q1 QX Mass 4/91

26 QX IBV History QX IBV was first reported in China circa 1995 (LX4- type virus) Currently it is the most frequently isolated IBV type in China In reported in Eastern Europe and Russia In reported in the Netherlands, Germany, France and Belgium Spread to England in 2007 Found in broilers, layers, breeders and backyard birds Causes Nephritis Vaccines are available Courtesy of Dr. Guillermo Zavala

27 Q1 IBV History 1996 to 1998 Q1(also T3 and J2) IBV first isolated in China 25 to 70 day-old layer chickens Respiratory disease/ Proventriculitis Q1, T3 and J2 are 98.9% similar Taiwan 2005 Chile 2009 Italy 2011 Colombia 2012 No vaccine is available at this time

Control using Vaccination IBV vaccines are given

hatchery and the field or by water in the field

28 Control using Vaccination IBV vaccines are given at 1-day of age in the hatchery and at 14 to 18 days of age in the field (broilers). The vaccine can be given by course spray in the hatchery and the field or by water in the field Multivalent vaccines are usually given in combination with NDV

29 Vaccines World wide Mass (H52 and H120) USA Mass (Mass 41), Conn, Ark-DPI, Holland (Mass) DE, GA98, (JMK, Fla) Europe Mass, 4/91 (793B) Australia Vac C, VicS, VacB2, VacB3 Netherlands Mass, D1466, D274, D1201

30 IBV Vaccine Efficacy The vaccine must have the same serotype as the challenge (field) virus (homologous protection) Little or no cross protection occurs between serotypes (heterologous protection) So how do we protect chickens against variant IBV strains?

can provide protection Some strains of IBV are particularly

31 Modified live vaccines and cross-protection Vaccinating with more than one serotype induces cross-reacting antibodies that can provide protection Some strains of IBV are particularly good at inducing these crossreacting antibodies (Ma5 & 4/91 or DE072)

Protection: Signs/Ciliostasis/Virus Detection Vaccination Challenge virus Day of hatch Day 14 GA 11 Mass 41 GA98 MA5 DE072 - / - / - A - / - / - - / - / - MA5 -- + / - / + - / - / + + / + / + --

32 Protection: Signs/Ciliostasis/Virus Detection Vaccination Challenge virus Day of hatch Day 14 GA 11 Mass 41 GA98 MA5 DE072 - / - / - A - / - / - - / - / - MA / - / + - / - / + + / + / + -- DE072 + / + / + + / + / + + / - / / + / + + / + / + + / + / + A Protection is based on: 90% protection from signs >50% protection against ciliostasis 90% protection against virus detection

33 Key Steps in the Control of IBV 1. Surveillance and identification of new variant viruses by sequence analysis is extremely important 2. Determine if new variants are pathogenic 3. Clinical case data and targeted surveys are used to determine how widespread the variant has become 4. Determine if current commercially available vaccines are efficacious 5. Development of a new vaccine for the variant if none of the commercial vaccines alone or in combination are efficacious

34 Thank you for your attention!

Worldwide perspective on Infectious Bronchitis. Ruth Bouwstra, DVM, PhD Turkey February 2017

Worldwide perspective on Infectious Bronchitis Ruth Bouwstra, DVM, PhD Turkey February 2017 Infectious bronchitis virus Corona Virus, a ssrna virus - Relatively high rate of mutations (0,0012 subst per